Influence And Mechanism Of Bortezomib In Combination With Sodium Valproate On RPMI8226 Cells

Objective: Multiple myeloma (MM) is a plasma-cell neoplasm. Until now, MM is an incurable malignancy. In recent years, thalidomide, lenalidomide and bortezomib as targeted therapy drugs are used to treat MM have brought encouraging progress.Bortezomib as a proteasome inhibitor, the status of treating newly-diagnosed, relapsed and refractory MM patients have been affirmed. How to preferably express the effect of bortezomib in the treatment of MM, more research is in progress. Studies have shown that bortezomib in combination with a variety of chemotherapy drugs have synergistic anti-apoptotic role in raising the overall response rate of multiple myeloma, and well tolerated. Also found that bortezomib have new molecular basis and mechanism in inducing MM cells apoptosis, which provide a theoretical basis for bortezomib in the treatment of other malignancies.In this study, multiple myeloma cell line RPMI8226 for the study, ompare the effect of using bortezomib in combination with using histone deacetylase inhibitor sodium valproate in the introduction of apoptosis in RPMI8226 cells with the effect of using bortezomib alone, or using VPA alone in the introduction of apoptosis, and discuss the two drugs synergistically induced apoptosis mechanism.Method:1 RPMI8226 cell culture and passageRPMI8226, a cell line derived from human multiple myeloma, were cultured in RPMI1640 medium, containing 20% inactivated fetal bovine serum (FBS), 100U/ml penicillin and 100μg/ml streptomycin sulfate, 10mmol / L of HEPES solution, at 37℃in an atmosphere of 5% CO2 , each passage 2 or 3 days. The experiments were performed when cells entered the logarithmic phase.2 Growth inhibition rate was measured with MTT assay methodRPMI8226 cells in logarithmic growth phase were inoculated into 96-well flat-bottomed culture plate adjust at a density of 6×104 cells/ml. Each-well was added to bortezomib at different concentrations, different concentrations of sodium valproate, or bortezomib in combination with different concentration of VPA. After the different incubation time, each well were added 20μl MTT, the supernatant were discarded after centrifugation, then add 200μl DMSO, shocking to restore dissolved purple crystals. The amount of converted MTT was quantified as value of absorbance(A) at 570nm test wavelength in an ELISA Reader. Then computed cell growth inhibition rate.3 The apoptosis rate was measured by flow cytometryRPMI8226 cells in logarithmic growth phase were planted in 6-well plates at a density of 2×10⁵ cells/ml, with 5ml cell suspension in each well. After cultured with bortezomib(20nM/ml), bortezomib(20nM/ml) in combination with different concentrations of sodium valproate, RPMI8226 cells were harvested, washed in 4℃PBS 2 times , and resuspended in 200μl Binging Buffer. Following addition of 10μl Annexin-FITC, gently mixing, 15min reaction at room temperature away from light, then added 300μl Binging Buffer and 5μl PI, immediately detected after mixing gently by flow cytometry, counting 10⁴ cells, CELLQuest software to analyze cell apoptosis.4 Detection of the expression HDACâ… Learn cells dealing with different concentrations of bortezomib, sodium valproate, valproate in combination with bortezomib, extracted total RNA. Measured OD₂₆₀/OD₂₈₀. Reverse transcribed cDNA. Toβ-actin expression as internal control, RT-PCR detection of HDACâ… expression under different culture conditions change.5 Statistical analysisSPSS18.0 software analysis, data as mean±standard deviation, correlation sample was analysed by non-parametric analysis of variance, P<0.05 indicated significant difference, P>0.05 for the difference was not statistically significant.Results:1 bortezomib, sodium valproate caused time- and dose- dependent growth inhibition of RPMI8226 cells. Cells were treated with bortezomib at the concentration of 10nmol/L, 20nmol/L, 30nmol/L, 40nmol/Lfor 24h, 48h, 72h. The cell proliferation inhibition rate was 3.1%, 7.37%, 12.37%, 17.88%; 4.92% , 27.28%, 37.69%, 41.91%; 14.6%, 41.92%, 59.14%, 64.02%. VPA treated cells at the concentration of 1mmol/L, 2mmol/L, 4mmol/L, 6mmol/L for 24h, 48h and 72h. The cell proliferation inhibition rate was respectively: 0.37%, 5.78%, 17.02%, 24.76%; 10.67%, 15.03%, 24.16%, 43.21%; 25.16%, 32.32%, 42.53%, 59.65%. Bortezomib (20nmol/L) combined sodium valproate (1mmol/L, 2mmol/L, 4mmol/L, 6mmoL/L) treated cells for 24h, 48h, 72h. The growth inhibition was 14.38%, 22.06%, 24.98 %, 34.25%; 34.63%, 44.61%, 55.74%, 64.63%; 45.84%, 50.44%, 57.19%, 63.86%. According to the effect of medication formula to work out Q, which demonstrate the two drugs in the valproate at the concentration≤4mmol/L, the role of a synergistic effect within 48h.2 RPMI8226 Cell apoptosis rate was detected by Annexin V/PI double staining assay.The result showed that the cell apoptotic rate was increased in a dose-dependent manner after cells treated with bortezomib (20nmol/L) in combination with sodium valproate (1mmol/L, 2mmol/L, 4mmol/L, 6mmol/L) for 48 hours. The apoptosis rate increased from 50.16±2.94% to 65.70±2.27%, which was significantly higher than the bortezomib (20nmol/L) group (P <0.05).3 RT-PCR results showed that bortezomib downregulated the expression of HDACâ… in a dose- and time- dependent manner. The decrease of the expression of HDACâ… of using bortezomib in combination with VPA is more obvious than singly using VPA.Conclusion:1 VPA can inhibit the proliferation of RPMI8226 cells, showing time-and dose-dependent manner. 2 bortezomib in combination with sodium valproate can enhance the proliferation inhibition and apoptosis of RPMI8226 cells, and two drugs compared to bortezomib alone or valproate have synergistic effect.3 Bortezomib in combination with VPA inhibited the growth of RPMI8226 cells have a synergistic effect, whose mechanism may be that bortezomib can reduce HDACâ… expression.